This proposal accomplishes the AREA program objectives of: 1) supporting meritorious research;2) exposing undergraduates to research;and 3) strengthening the research environment in non-research intensive universities. The general goal of this research is to elucidate the mechanism of polyhydroxyalkanoate inclusion biogenesis. Polyhydroxyalkanoates (PHAs) are bacterial polymers that are synthesized in inclusions when carbon levels are high and another essential nutrient, such as nitrogen is limited. There is considerable commercial interest in PHAs because they comprise a family of polyesters that can be formed into plastics that are biodegradable. Electron microscopy studies have been unable to resolve the structure of PHA inclusions and this has inhibited movement toward a cohesive model of inclusion biogenesis. Employing atomic force microscopy, we have determined that there are three layers of structure, an outer envelope that is the thickness of a membrane bilayer, a middle network layer, and an underlying crystalline lamellar layer. Genetic studies have indicated that the middle network is comprised at least partially of PhaP and that PhaP is likely to be translocated to the periplasm. Thus, it would appear that inclusion biogenesis may occur by movement of protein and/or proteins to the periplasm and budding through the cytoplasmic membrane into the cytoplasm, facilitating the acquisition of the cytoplasmic membrane as an envelope. The goal of this research is to prove or disprove this supposition. The specific aims of the research are: 1) definitively prove periplasmic localization of PhaP via fluorescence localization and Western blot analyses of subcellular fractions, 2) demonstrate that the inclusion envelope is derived from the cytoplasmic membrane by proteomic analysis, and 3) characterize proteins that bind transiently and permanently to PhaP in hopes of elucidating the mechanism of inclusion biogenesis. Ultimately, the goal of the research is to enlarge our knowledge of inclusion biogenesis to the point that this process can be controlled and utilized for medical applications. For instance, it could be envisioned that instead of polymer being inserted into the inclusion, bioactive compounds could be inserted, making the inclusion into a drug delivery vehicle. Morehead State University has recently embarked upon a process whereby undergraduate research is emphasized and faculty members are encouraged to become teacher/scholars. Significant resources have been allocated to this goal. This project supports this mission and will enhance the research environment at MSU by providing undergraduate students with numerous opportunities to learn the fundamentals of biomedical research while conducting research that will enlarge our knowledge of prokaryotic processes. PUBLIC HEALTH RELEVANCE: Using atomic force microscopy we have resolved the structural arrangement of polyhydroxyalkanoate inclusions and this has led to a preliminary model for inclusion biogenesis. The purpose of this research is to test this preliminary model in hopes of adapting the strategy of biogenesis for medical applications in which inclusions could be used as drug delivery vehicles.